The hypothesis of this proposal is that the biosynthesis of crustacean chitin occurs in 3 stages: (a) synthesis of a chitin oligosaccharide on a lipid-linked intermediate, i.e. dolichol pyrophosphate; (b) transfer of the chitin oligosaccharide from the lipid to a protein acceptor forming a chitoprotein primer; and (c) the extension of primer oligosaccharides by chitin synthetase. The goal of the present proposal is to examine the pathway of chitin synthesis within the crustacean epithelial cell and to study the mechanism of assembly of the chitin-protein complex into the crustacean exoskeleton. The model systems for this research include larval stages of two crustaceans, Artemia salina and Penaeus setiferus; the results obtained in this study will be compared to existing data on chitin synthesis in fungal systems, e.g. Saccharomyces cerevisiae. In vitro and in vivo assay procedures which have been developed during the previous grant period, will be used to monitor the synthesis of lipid-linked oligosaccharides, chitoproteins and macronuclear chitin. The proposed research will include: (1) Isolation and partial purification of cellular organelles involved in crustacean chitin synthesis, e.g. rough endoplasmic reticulum, golgi apparatus and plasma membrane; (2) assay of lipid-linked oligosaccharide, chitoprotein and macromolecular chitin synthesis in each of these fractions; (3) electron microscopic studies to examine epithelial cells and isolated organelles and visualize the synthesis and deposition of chitin; (4) preparation of polyclonal antibodies against chitoprotein product to be used in assay procedures, SDS-PAGE analysis of synthesis reactions and electron microscope studies; (5) studies on inhibitors of chitin assembly, including the eafects of monensin, calcofluor white, flavomycin, tinopal, vancomycin and amphotericin B. (6) Studies on specific enzymes involved in the three step synthesis process: (a) the oligosaccharide transferase: its specificity and inhibitors; (b) chitin synthetase: its solubilization and partial purification plus comparison to its fungal counterparts; and (c) tanning enzymes: does diflubenzuron inhibit the crosslinking of the crustacean cuticle?